1. Field of the Invention
The present invention relates generally to the field of recombinant DNA vectors, and particularly concerns vectors useful for producing a desired protein of interest in an insect cell that has modifications similar to the same protein produced in mammalian cells. More particularly, it concerns recombinant baculovirus vectors that are used to infect or stably transform insect cells, directing the production of oligosaccharide processing enzymes, other protein modification enzymes and proteins which aid in proper protein folding, thereby obtaining the desired protein. The invention also concerns insect cells with stably integrated protein modification enzymes, and methods utilizing the vectors, viruses and cells disclosed herein.
2. Description of Related Art
One of the major benefits provided by recombinant DNA technology is the ability to express cloned genes in a heterologous host, which facilitates the isolation of large amounts of foreign gene products for further study or direct practical applications. Bacterial systems typically provide high expression levels, but lack eucaryotic protein processing capabilities. Many biomedically important proteins are processed, and the lack of processing can alter their folding, transport, stability and/or function (Welply, 1991). Also, the foreign gene product is often deposited as an insoluble inclusion body. Mammalian systems can provide protein processing, but expression levels are much lower and mammalian cells are much more expensive to cultivate. An ideal system would combine high expression levels, proper protein processing, and still be relatively inexpensive.
Baculoviruses are DNA-containing viruses that infect insects or other invertebrates (Adams and McClintock 1991). Baculovirus vectors usually provide high levels of foreign gene expression, and the insect cell hosts have some eucaryotic protein processing capabilities. Also, while insect cells remain more expensive to cultivate than bacteria, recent developments have significantly reduced the cost of producing foreign gene products in this system. Based on these properties, the baculovirus-insect cell system is a widely used tool for the production of foreign gene products, particularly eucaryotic proteins that must be co- and post-translationally processed (Summers and Smith, 1987, Luckow and Summers, 1988; Miller, 1988; O""Reilly et al., 1992).
However, a major limitation of using the baculovirus-insect cell system for recombinant glycoprotein production is that the N-glycosylation pathway in insect cells differs from the pathway found in higher eukaryotes (Jarvis and Summers, 1992; Kornfeld and Kornfeld, 1985). This is a significant drawback as there is increasing evidence that proper glycosylation imparts important functions to many eukaryotic proteins (Welply, 1991).
Most of the information on the N-glycosylation pathway in insect cells has come from structural studies on foreign glycoproteins expressed in baculovirus-infected cell lines or in larvae (reviewed by Jarvis and Summers, 1992; O""Reilly et al., 1992; Jarvis, 1993a). These studies have demonstrated that insect cells have processing glucosidases and mannosidases which convert high mannose oligosaccharides to trimmed structures with as few as three mannose residues. Several lines of evidence indicate that these cells also have a fucosyltransferase that can add fucose to the core Asn-linked GlcNAc residue (Staudacher et al., 1992).
However, mammalian cells extend such trimmed oligosaccharide structures by adding N-acetylglucosamine, galactose, fucose, and sialic acid residues to produce a complex biantennary structure containing penultimate galactose and terminal sialic acid residues (Kornfeld and Kornfeld, 1985; Paulson and Colley, 1989; Moremen et al., 1994). Insect cells generally do not produce these extended complex structures, indicating that the requisite processing activities are either absent or too low to be generally effective in these cells. This limits the current usefulness of insect cells. Although some recent studies indicate that insect cell lines can produce glycoproteins with certain terminal glycosylation patterns more similar to those found in higher eukaryotes (Kubelka et al., 1994; Ackermann et al., 1995, Ogonah, et al., 1996, Davidson et al., 1990; Davidson and Castellino, 1991a), the vast majority of recombinant proteins produced in insect cells lack these structures.
The present invention overcomes the drawbacks in the prior art by providing new and improved baculoviral expression vectors, insect cell lines, compositions and various methods of use. The invention first provides a baculovirus expression vector characterized as either: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene operatively positioned under the control of and in frame with a promoter; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene operatively positioned under the control of a baculoviral immediate early, delayed early, early or late promoter.
Recombinant vectors for important aspects of the present invention. The term xe2x80x9cexpression vector or constructxe2x80x9d means any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a RNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid, for example, to generate antisense or ribozyme constructs. It will naturally be understood that the transcriptional units each comprise the appropriate transcription and translation initiation and termination signals, such as ATG start signals, and are positioned in the proper orientation to allow transcription of the gene.
Where the present invention comprises the vector of part (a) above, it will be understood that the vector may a vantageously further include a third, fourth, fifth, sixth, seventh, eighth and/or a ninth glycosylation enzyme transcriptional unit, the third, fourth, fifth, sixth, seventh, eighth and/or a ninth transcriptional unit comprising a structural gene for a third, fourth, fifth, sixth, seventh, eighth and/or a ninth oligosaccharide processing enzyme, operatively positioned under the control of a promoter.
Equally, where the present invention comprises the vector of part (b) above, the vector may also further include a second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth glycosylation enzyme transcriptional unit, the second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth transcriptional unit comprising a structural gene for a second, third, fourth, fifth, sixth, seventh, eighth and/or a ninth oligosaccharide processing enzyme, operatively positioned under the control of a promoter.
Exemplary oligosaccharide processing enzymes for use in the invention include, but are not limited to xcex1-glucosidases, including xcex1-glucosidase I and xcex1-glucosidase I, xcex1-mannosidases, such as xcex1-mannosidase I and xcex1-mannosidase II, N-acetylglucosaminyltransferases, including, but not limited to N-acetylglucosaminyltransferase and N-acetylglucosaminyltransferase II, fucosyltransferases, galactosyltransferases and sialyltransferases. The oligosaccharide processing enzymes contemplated for use in the present invention include, but are not limited to, the extensive list provided herein below in Table 1.
The oligosaccharide processing enzymes may be used individually, or in any combination. In certain preferred embodiments, the oligosaccharide processing enzyme will be a galactosyltransferase. In other preferred embodiments, a galactosyltransferase will be used in combination with sialyltransferase. In further preferred embodiments, a galactosyltransferase will and a sialyltransferase will be used in conjunction with N-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase II.
Particularly useful vectors are contemplated to be those vectors in which the coding portion of a DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the transcriptional control of a promoter. A xe2x80x9cpromoterxe2x80x9d refers to a DNA sequence recognized y the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases xe2x80x9coperatively positionedxe2x80x9d, xe2x80x9cunder controlxe2x80x9d or xe2x80x9cunder transcriptional controlxe2x80x9d means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. Promoters for use in the present invention include insect cell promoters, viral promoters, and preferably baculovirus promoters, such as IE1, IEN (IE2) IE0, 39K, gp64, DA26, ETL, 35K, capsid (p39) p10 and the polyhedrin promoter.
The viral promoters for use in the present invention may be obtained from the viral DNA of Autographa californica NPV, Trichoplusia ni NPV, Rachipulsia ou NPV, Orgyia pseudosugata NPV, Bombyx mori NPV, Heliothis zea NPV, Spodoptera exigua NPV or Galleria mellonella NPV.
Additionally, the current invention provides for the use of enhancer elements operatively positioned to enhance expression of the transcriptional units. All eukaryotic enhancers are contemplated for use in the present invention, with preferred enhancers being the baculovirus enhancers hr1, hr2, hr3, hr4 and/or hr5, with the viral DNA of Autographa californica NPV, Trichoplusia ni NPV, Rachipulsia ou NPV, Orgyia pseudosugata NPV, Bombyx mori NPV, Heliothis zea NPV, Spodoptera exigua NPV or Galleria mellonella NPV being the preferred sources for the baculoviral enhancer elements.
Additional proteins that modify or stabilize proteins are contemplated for use in the present invention, either alone, in conjunction with the oligosaccharide processing enzymes. Certain embodiments of the present invention provide a structural gene encoding a modification protein including, but not limited to, a protein kinase, a protein methylase, and proteins involved in acylation, acetylation and/or amidation of proteins, operatively positioned under the control of a promoter. Other embodiments of the present invention provide a structural gene encoding a protein involved in the stabilization or proper folding of proteins, including, but not limited to, protein disulphide isomerase, peptidyl prolyl cis-trans isomerase and/or a chaperone protein, operatively positioned under the control of a promoter referred from this class of proteins is a structural gene for BiP/GRP78, and particularly preferred is BiP/GRP78 from an insect cell.
Further embodiments of the present invention include a structural gene encoding a screenable or selectable marker protein, operatively positioned under the control of a promoter. Exemplary of these screenable marker proteins are xcex2-galactosidase, chloramphenicol acetyltransferase, xcex2-glucuronidase, luciferase and green fluorescent protein. Preferred for use in the present invention are selectable marker proteins, including, but not limited to, antibiotic or toxin resistance genes such as neomycin resistance, hygromycin resistance and dihydrofolate reductase, which confers resistance to methotrexate.
In particular aspects of the present invention, the vectors further comprise a baculovirus structural gene, with gp64, p10 and/or polyhedrin being preferred examples. Further embodiments of the present invention include a cloning restriction site, optionally, and preferably, for insertion of one or more heterologous coding regions or genes that encode one or more proteins or polypeptides to be expressed. In particularly preferred embodiments, the cloning restriction site comprises a DNA insert including a multiple cloning cassette.
Certain embodiments of the present invention include at least one heterologous structural gene encoding a selected protein, the gene operatively positioned under the control of and in frame with, a promoter. Preferred are baculoviral promoters, more preferred are very late baculoviral promoters, and particularly preferred are the polyhedrin and/or p10 promoter. Alternatively the promoter is a promoter naturally associated with the heterologous structural gene.
In additional embodiments of the present invention, the vectors comprise a structural gene encoding an insecticidal protein. Insecticidal proteins preferred for use include, but are not limited to, Bacillus thuringiensis crystal toxins, protease inhibitors, lectins, chitinases, proteases, insect-specific neurotoxins and trypsin inhibitors. Particularly preferred are juvenile hormone esterase and the insect-specific toxins Androctonus australis toxin (AaIT) and Leiurus quinquestriatus hebraeus toxin (LqhIT2).
The present invention additionally provides a 5xe2x80x2 end flanking baculovirus viral DNA and a 3xe2x80x2 end flanking baculovirus viral DNA, allowing recombination of the transcriptional units into the baculovirus genome, thereby replacing a portion of the baculovirus genome. Preferred portions of the baculovirus genome for replacement are the gp64, p10 and/or polyhedrin loci.
In certain preferred embodiments of the present invention, the first oligosaccharide processing enzyme structural gene encodes a galactosyltransferase and the second oligosaccharide processing enzyme structural gene encodes a sialyltransferase. In preferred embodiments, at least one of the first or second oligosaccharide processing enzyme structural genes is operatively positioned under the control of a baculovirus promoter. Preferred baculovirus promoters are baculovirus immediate early, delayed early and/or early promoters. Further preferred uses of the current invention provide the structural gene encoding N-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase II, operatively positioned under the control of the above promoters. Particularly preferred embodiments further include a baculoviral enhancer. Preferred embodiments provide these components irrespective of the particular vector construct used. In certain embodiments of the present invention, the vectors are encapsulated within a baculovirus.
Thus in particular aspects the present invention provides a baculovirus expression vector comprising a gene encoding an oligosaccharide processing enzyme, a chaperone protein, a protein stabilization protein and/or another type of protein modification enzyme operatively positioned under the control of a promoter, a baculoviral structural gene, a 5xe2x80x2 end flanking baculovirus viral DNA, a 3xe2x80x2 end flanking baculovirus viral DNA, and a structural gene encoding a chaperone protein operatively positioned under the control of a promoter.
In further embodiments, the gene encoding the oligosaccharide processing enzyme is operatively positioned under the control of a promoter and a baculoviral enhancer. Additional embodiments include a structural gene encoding a screenable or selectable marker protein operatively positioned under the control of a promoter. In preferred embodiments, the gene encoding the selectable marker protein encodes an antibiotic or toxin resistance gene. Particularly preferred embodiments include a DNA insert including a multiple cloning site. Exemplary embodiments further include a heterologous structural gene inserted into the multiple cloning site.
The present invention also provides a baculovirus particle comprising a baculovirus expression vector characterized as: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene operatively positioned under the control of a promoter; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene operatively positioned under the control of a baculoviral immediate early, delayed early, early or late promoter.
An embodiment of the present invention is an insecticidal composition comprising a population of baculovirus particles, the baculovirus particles comprising a baculovirus expression vector characterized as: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene operatively positioned under the control of a promoter; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene operatively positioned under the control of a baculoviral immediate early, delayed early, early or late promoter.
An additional embodiment of the present invention is an insect cell comprising a baculovirus expression vector characterized as: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene operatively positioned under the control of a promoter; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene operatively positioned under the control of a baculoviral immediate early, delayed early, early or late promoter.
The insect cell may have integrated into its genome one or more functional units from the baculovirus expression vector. Thus, in further embodiments of the current invention, the insect cell is a stably transformed insect cell line or clone that expresses or continually expresses at least a first glycosylation enzyme not normally expressed, or not normally expressed at sufficiently functional levels, in the natural insect cell. Preferred insect cells are Lepidopteran insect cells, and particularly preferred are Spodoptera frugiperda, Bombyx mori, Heliothis virescens, Heliothis zea, Mamestra brassicas, Estigmene acrea or Trichoplusia ni insect cells.
In certain aspects, the vectors of the present invention are contemplated for use in the preparation of a recombinant baculovirus. Thus, the present invention provides a baculovirus containing any of the baculovirus expression vectors disclosed herein. The present invention also provides for the use of the vectors of the instant invention in the preparation of a recombinant baculovirus. In further aspects, the invention provides a population of baculovirus particles containing any of the instant baculovirus expression vectors. In other preferred embodiments, a baculovirus containing any of the baculovirus expression vectors disclosed herein are contemplated for use in the preparation of an insecticidal formulation. Thus, the present invention also provides for the use of a baculovirus containing any of the baculovirus expression vectors of the present invention in the preparation of an insecticidal formulation.
Also provided by the present invention is a method for metabolically engineering an insect cell, comprising providing to the cell at least a first baculovirus expression vector characterized as: (a) comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene positioned under the control of a promoter operative in the insect cell; or (b) comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene positioned under the control of a baculoviral immediate early, delayed early, early or late promoter operative in the insect cell.
The invention further provides a method for metabolically engineering an insect cell, comprising providing to an insect any of the baculovirus expression vectors disclosed herein. Also provided are baculovirus expression vectors for use in metabolically engineering an insect cell. Thus, the present invention provides for the use of any of the disclosed baculovirus expression vectors in the metabolic engineering an insect cell.
In further embodiments, the insect cell line is provided with the first baculovirus expression vector by infection. Alternatively, the insect cell line is provided with the first baculovirus expression vector by transfection. In an additional method of practicing the present invention, the first baculovirus expression vector is maintained extrachromosomally in the insect cell to provide an insect cell that transiently expresses the oligosaccharide processing enzyme or enzymes. In an alternate method of the current invention, the first baculovirus expression vector stably integrates into the genome of the insect cell line to provide a stably transformed insect cell that continuously expresses the oligosaccharide processing enzyme or enzymes.
The present invention thus further provides an insect cell containing any of the baculovirus expression vectors disclosed herein. In further aspects, a baculovirus containing any of the disclosed baculovirus expression vectors are provided for use in the preparation of an engineered insect cell. Thus, the present invention provides for the use of a baculovirus containing any of the disclosed baculovirus expression vectors in the preparation of an engineered insect cell. The invention also provides an engineered insect cell that expresses at least a first heterologous oligosaccharide processing enzyme. A further embodiment of the instant invention is an engineered insect cell that expresses at least a first heterologous oligosaccharide processing enzyme and at least a first heterologous gene that encodes a selected protein.
In a preferred method, the first baculovirus expression vector expresses a galactosyltransferase and a sialyltransferase oligosaccharide processing enzyme. Particularly preferred is where the first baculovirus expression vector further expresses N-acetylglucosaminyltransferase II and N-acetylglucosaminyltransferase II. In certain methods of the present invention, the insect cell is a Lepidopteran insect cell. In preferred methods, the insect cell is a cultured insect cell, or in the alternative is housed within a living insect.
In additional methods of the present invention, the insect cell is further provided with a heterologous structural gene that expresses a selected protein in the insect cell. In particular methods, the heterologous structural gene is provided to the insect cell by means of a baculovirus expression vector. In certain methods of the present invention, the heterologous structural gene is comprised within the first baculovirus expression vector. In additional methods, the heterologous structural gene is comprised within a second baculovirus expression vector that is provided to the insect cell.
The present invention provides a method for producing a selected protein in an insect cell, comprising preparing an engineered insect cell that expresses at least a first heterologous oligosaccharide processing enzyme and expressing in the engineered insect cell a heterologous gene that encodes the selected protein. Additional methods comprise collecting the selected protein expressed by the cell.
The present invention also provides a method for producing a selected protein in an insect cell, comprising providing to an insect any of the baculovirus expression vectors of the instant invention and an expressible nucleic acid segment encoding said selected protein. Additionally, baculovirus expression vectors for use in producing an oligosaccharide-containing selected protein in an insect cell are provided. Thus, the present invention provides for the use of any of the disclosed baculovirus expression vectors in the production of an oligosaccharide-containing selected protein in an insect cell. In other embodiments, baculovirus expression vectors are provided for use in preparing a formulation for use in producing an oligosaccharide-containing selected protein in an insect cell. Therefore, the present invention additionally provides for the use of any of the instant baculovirus expression vectors in the preparation of a formulation for use in producing an oligosaccharide-containing selected protein in an insect cell.
The present invention also provides a method for producing a selected protein in an insect cell, comprising providing to the insect cell at least a first baculovirus expression vector that expresses at least a first oligosaccharide processing enzyme in the insect cell, and further providing to the cell a heterologous gene that expresses the selected protein in the insect cell. The method may be characterized as comprising the steps of preparing a first baculovirus expression vector in which an oligosaccharide processing enzyme gene is positioned under the control of a promoter operative in the insect cell, introducing the baculovirus expression vector into an insect cell, and maintaining the insect cell under conditions effective to allow expression of the encoded selected protein. Preferred methods of the present invention include collecting the expressed selected protein and purifying the expressed protein away from total cell components.
In additional methods, the heterologous structural gene is provided to the insect cell by means of a baculovirus expression vector. In alternate methods of the present invention, the heterologous structural gene is comprised within the first baculovirus expression vector, or within a second baculovirus expression vector that is provided to the insect cell.
In a preferred method of the present invention, the first baculovirus expression vector expresses galactosyltransferase and sialyltransferase oligosaccharide processing enzymes. In additional methods, the first baculovirus expression vector further expresses N-acetylglucosaminyltransferase I and N-acetylglucosaminyltransferase II enzymes.
The present invention provides a selected recombinant protein polypeptide prepared by expressing a heterologous gene encoding the protein or polypeptide in a recombinant insect cell, as disclosed herein, and purifying the expressed protein or polypeptide away from total recombinant host cell components.
The present invention further provides a method for producing a selected protein in an insect cell, comprising the steps of creating an engineered insect cell by providing to an insect cell at least a first baculovirus expression vector characterized as comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene positioned under the control of a promoter operative in the insect cell, or comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene positioned under the control of a baculoviral immediate early, delayed early, early or late promoter operative in the insect cell, and expressing in the engineered insect cell a heterologous gene that expresses the selected protein.
The present invention also provides a method for producing a selected protein in an insect cell, comprising preparing a stably transformed insect cell that expresses at least a first oligosaccharide processing enzyme and infecting the stably transformed cell with a baculovirus comprising an expression vector that comprises a heterologous gene that expresses the selected protein in the insect cell.
Additionally, the present invention provides a method for killing an insect cell, comprising contacting the insect cell with at least a first baculovirus expression vector that expresses at least a first oligosaccharide processing enzyme in the insect cell. In a preferred method, the baculovirus expression vector is encapsulated within a baculovirus. Thus in certain embodiments, the invention provides an insect containing one or more of the baculovirus expression vectors disclosed herein.
These methods are generally based upon the classical use of baculovirus alone to kill insects. As the virus life cycle requires the virus to infect an insect cell, to reproduce and ultimately to kill the host insect cell and release new viruses, baculovirus alone is insecticidal. The narrow host cell specificity means that the use of baculovirus in the environment is not generally deleterious to cells, plants and animals other than insect cells.
However, the insect target cells can adapt to be less-sensitive to baculovirus infection. This process is believed to include, at least in part, recognition and activity of glycosylated proteins. Therefore, the new baculovirus expression vectors of the present invention that express at least one oligosaccharide processing enzyme, the enzyme not normally present or significantly present in insect cells, will function to change the glycosylation pattern of the proteins in the cells, which should hamper the ability of the cells to become resistant to baculovirus.
In further methods, the insect cell is housed within a living insect. That is, the methods are applicable to insect cells in culture, and to whole, live insects. In additional methods of the present invention, the baculovirus expression vector is characterized as comprising at least a first and a second glycosylation enzyme transcriptional unit, the transcriptional units comprising a first and a second structural gene encoding a first and a second oligosaccharide processing enzyme, each gene positioned under the control of a promoter operative in the insect cell, or comprising at least a first glycosylation enzyme transcriptional unit, the transcriptional unit comprising a structural gene encoding an oligosaccharide processing enzyme, the gene positioned under the control of a baculoviral immediate early, delayed early, early or late promoter operative in the insect cell.
In preferred methods of the present invention, the baculovirus expression vector further expresses an insecticidal protein in the insect cell. In particularly preferred methods, the insecticidal protein is Bacillus thuringiensis crystal toxin, a protease inhibitor, a protease, an insect-specific neurotoxin or other toxins, a lectin, a chitinase, juvenile hormone esterase or a trypsin inhibitor insecticidal protein. In exemplary examples of the present invention, the insecticidal proteins are lectins such as wheat germ agglutinin, rice lectin or stinging nettle lectin, Bacillus thuringiensis crystal toxin genes such as CrylA (b) or CrylA(c), or insect-specific toxins such as AaIT or LqhIT2.
In additional methods of the present invention, the vector expresses a glycosylatable insecticidal protein and the oligosaccharide processing enzyme functions to modify the glycosylation pattern of the insecticidal protein, wherein the vector expresses a glycosylatable insecticidal protein that requires a defined glycosylation pattern to achieve significant insecticidal activity and wherein the co-expressed oligosaccharide processing enzyme functions to modify the glycosylation pattern of the insecticidal protein expressed in the insect cell sufficiently to increase its insecticidal activity, i.e., wherein the oligosaccharide processing enzyme modifies the glycosylation pattern of the insecticidal protein by adding one or more terminal glycosyl residues that are not normally added to the protein when the protein alone is expressed in the insect cell.
In preferred methods of the present invention, the vector expresses the insecticidal protein juvenile hormone esterase, AaIT and/or LqhIT2 and the oligosaccharide processing enzyme galactosyltransferase and/or sialyltransferase. In other aspects, the vector expresses a chaperone protein, such as BiP/GRP78, in conjunction with an insecticidal protein, such as the insect-specific toxins AaIT and/or LqhIT2.
The present invention further provides DNA segments that comprise an isolated insect xcex1-mannosidase I and/or xcex1-mannosidase II gene or cDNA, as may be isolated from lepidopteran insect cells, such as Sf9, High Five or Ea cells. The xcex1-mannosidase II or xcex1-mannosidase I genes or cDNAs are DNA segments that comprise gene sequences, or coding regions, that encode xcex1-mannosidase II or xcex1-mannosidase I proteins, polypeptides or peptides.
In certain embodiments, the xcex1-mannosidase II genes and cDNAs will include a contiguous nucleic acid sequence that encodes an xcex1-mannosidase II protein, polypeptide or peptide that comprises a contiguous amino acid sequence from the amino acid sequence of SEQ ID NO:4. In further embodiments, the xcex1-mannosidase II genes and cDNAs will include a contiguous nucleic acid sequence that corresponds to a contiguous nucleic acid sequence from the nucleic acid sequence of SEQ ID NO:3, and preferably, from the open reading frame thereof. Nucleic acid sequences comprising the complement of SEQ ID NO:3 are also provided.
In certain other embodiments, the xcex1-mannosidase I genes and cDNAs will include a contiguous nucleic acid sequence that encodes an xcex1-mannosidase I protein, polypeptide or peptide that comprises a contiguous amino acid sequence from the amino acid sequence of SEQ ID NO:2. In further other embodiments, the xcex1-mannosidase I genes and cDNAs will include a contiguous nucleic acid sequence that corresponds to a contiguous nucleic acid sequence from the nucleic acid sequence of SEQ ID NO:1, and preferably, from the open reading frame thereof. Nucleic acid sequences comprising the complement of SEQ ID NO:1 are also provided.
It will be understood that nucleic acid segments of from 14 to about 10,000 nucleotides in length that hybridize to the nucleic acid segment of SEQ ID NO:3 or SEQ ID NO:1, or the complement thereof, under standard or high stringency hybridization conditions are also included within the present invention. Where the insect xcex1-mannosidase II and I gene constructs encode xcex1-mannosidase II and I polypeptides or peptides, contiguous amino acid sequences of from about 15 to about 50, or from about 15 to about 30 amino acids in length are contemplated. These may be used in, e.g., immunization to generate antibodies to xcex1-mannosidase II and I.
Insect xcex1-mannosidase II and I gene constructs encoding full length proteins will generally be preferred in embodiments concerning the production of active proteins. The insect xcex1-mannosidase II and I genes and cDNAs of the present invention may be positioned under the control of a promoter, preferably a promoter that directs the expression of the xcex1-mannosidase II or I proteins in an insect cell. As such, the present invention also provides recombinant vectors comprising insect xcex1-mannosidase II and I genes and/or cDNAs that express xcex1-mannosidase II and/or I proteins. The present invention also provides recombinant host cells, preferably insect cells, comprising or incorporating DNA segments that comprise isolated xcex1-mannosidase II and/or I genes or cDNAs that encode insect xcex1-mannosidase II and/or I proteins. The genes or cDNAs are preferably introduced by means of a recombinant vector and the cell preferably expresses the vector.
The invention thus further provides methods of using a DNA segment that includes an isolated insect xcex1-mannosidase II and/or I gene or cDNA that encodes an xcex1-mannosidase II and/or I protein, comprising the steps of preparing a recombinant vector in which an insect xcex1-mannosidase II and/or I-encoding DNA segment is positioned under the control of a promoter, introducing the recombinant vector into a recombinant host cell, preferably an insect cell, culturing the recombinant host cell under conditions effective to allow expression of an insect encoded xcex1-mannosidase II and/or I protein or peptide, and collecting the expressed xcex1-mannosidase II and/or I protein or peptide.
Recombinant insect xcex1-mannosidase II and/or I protein, polypeptide or peptide compositions prepared by expressing xcex1-mannosidase II and/or I in a recombinant host cell are also provided, with preferred compositions comprising xcex1-mannosidase II and/or I polypeptide or peptide composition purified, or substantially, purified away from total recombinant host cell components. Antibodies that specifically bind to, i.e., have immunospecificty for insect xcex1-mannosidase II and/or I proteins, polypeptides or peptides are also provided.
Methods for detecting insect xcex1-mannosidase II and/or I sequences in a sample are also provided, which comprise obtaining sample nucleic acids from a sample suspected of containing insect xcex1-mannosidase II and/or I, contacting the sample nucleic acids with a nucleic acid segment that encodes an insect xcex1-mannosidase II and/or I protein or peptide under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting the hybridized complementary nucleic acids thus formed.
In situ hybridization, Northern and Southern blotting are contemplated. Also, pairs of nucleic acid primers that hybridize to distant sequences from insect xcex1-mannosidase II and/or I nucleic acid sequences may be used, wherein the primers are capable of amplifying an insect xcex1-mannosidase II and/or I nucleic acid segment when used in conjunction with a polymerase chain reaction. In such a polymerase chain reaction, amplification products are created and the amplification products thus formed are then detected. Thus, nucleic acid detection kits are provided which comprise, in suitable container means, one or more isolated insect xcex1-mannosidase II and/or I nucleic acid segments and, optionally, detection reagents.